Several lines of experimental evidence indicate that disruption of certain aspects of intra-cellular cholesterol homeostasis in various cell types (e.g. macrophage, ?-cell) can lead to pathological processes preceding type 2 diabetes mellitus (T2DM) and atherosclerotic vascular disease (ASCVD). Our recent transcriptomic study of purified human monocytes corroborates these findings, and specifically identifies a co-expressed cholesterol metabolism transcriptional network (CMTN) whose alteration is significantly associated with T2DM and coronary artery calcification (CAC, a subclinical ASCVD measure). This network includes 11 genes involved in coordinated up-regulation of cholesterol uptake and synthesis, and down-regulation of cholesterol efflux - a molecular profile expected to increase intracellular cholesterol. To translate these intriguing observations into meaningful improvements in human health, our goal is to comprehensively characterize the epigenetic regulators of this network of genes in human monocytes, and to investigate how this network and its regulatory factors relate to intra-cellular cholesterol in the monocytes and to the development of T2DM and ASCVD. Our principle focus will be on epigenetic regulation of this network by microRNAs (miRNAs). It is already well established via in vitro and animal models that one specific miRNA (miR-33) plays a critical role in cholesterol homeostasis in concert with its co-transcribed host gene, SREBP2. Our pilot data from 373 human monocyte samples indicate that intra-cellular levels of miR-33a is associated with expression of the entire gene network of interest in this proposal and with prevalent T2DM in the cell donors. We also identified several other promising miRNA candidates associated with expression of the gene network. Based on these preliminary data, and taking advantage of the well-phenotyped Multi-Ethnic Study of Atherosclerosis (MESA) cohort with existing genomic data, DNA methylomic and transcriptomic data on 1,264 monocyte samples, and miRNA sequencing data in a subset of 373 monocyte samples, we now proposes to additionally quantify miRNAs in the remaining 891 monocyte samples using next generation sequencing to achieve the following specific aims: 1) To characterize the relationship between miRNAs and the CMTN in 1,264 MESA monocyte samples; 2) To establish the association of miRNAs with T2DM and CAC in the 1,264 MESA participants; 3) To replicate miRNA associations with the most compelling evidence in an independent set of 562 MESA participants; and 4) To validate the functional consequences of the CMTN alterations and associated-miRNAs, using ex-vivo cultured human monocytes. The integration of genetic, epigenetic, transcriptional, and clinical data along with the ex-vivo experimental studies may provide novel mechanistic insights concerning the regulation of cholesterol metabolism and susceptibility to T2DM and ASCVD and lead to new strategies for prevention and treatment of T2DM and ASCVD.